A Dopable Bioink for Augmented Tissue Engineering in Craniofacial Reconstruction, Innovative Pipeline for Drug Design and Selective Delivery Through Functionalized Polymeric Nanoparticles.

To develop a mouldable biosynthetic collagen-based polymer matrix, commercial gelatin-methacrylamide (GelMA)-based hydrogels will be biochemically modified to finely tune their pseudo-plasticity and yield stress, and functionalized to implement drug delivery for improved biological properties. The GelMA will be chemically enriched with: adhesive molecules (e.g. alginate-based or ion releasing inorganic compounds), if needed, to increase the adhesion to implantable materials used in craniofacial reconstruction, thiol-based non- cleavage-type photoinitiators (e.g. eosinY combined with triethanolamine and vinyl caprolactam) to enable visible light-activated crosslinking and minimize the safety concerns of UV light. Photo-crosslinking will be achieved through a portable visible light device (420-480 nm) to induce the jellification of the bioink. The physical properties (morphology, viscoelastic properties, stiffness, resistance to an applied stress) of the different GelMA compounds will be analyzed according to standardized procedures. The adhesive properties of the GelMA will be measured by lap-shear strength tests in dry conditions. Once identified GelMA compounds showing the highest biological features, this will be endowed with functionalized Poly (lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs).

To this aim, biocompatible PLGA-Polyethylene glycol (PEG) NPs will be synthesized and functionalized with bis-sulfone for binding targeting moieties on the surface. The synthesis involves PLGA activation, PLGA-PEG conjugation, bis-sulfone activation, and PLGA-PEG-bis-sulfone conjugation. The PLGA/PEG ratio will be adjusted for the hydrophobicity/hydrophilicity payload. Bis-sulfone is used for selective and efficient PEGylation of protein's disulfide bond or to conjugate His-tagged protein/peptides according to standardized methods to implement either specific cell targeting or antimicrobial agents. High performance liquid chromatography (HPLC) will be used to validate the biosynthetic reaction. Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), and Scanning electron microscopy (SEM) will define the morphological and ultrastructural properties of the final construct. Surface Plasmon Resonance will validate target recognition. Then, GelMA-NP hybrid compound assembly will be achieved through extrusion 3D printing according to standardized production pipelines. The hydrogel and the NP suspension will be dispensed through separate nozzles, in 2D and 3D patterns with 100-1000 nm feature sizes, under pressure and temperature control. Scalar NP concentrations will be used and then evaluated.

NP integrity and release dynamics of these from GelMA will be studied by submerging the GelMA-NPs loaded with fluorescent compound with a bio-mimetic cell growth medium and quantifying the NPs released in the supernatant in time course experiments. NP quantification will be performed using UV-Vis Spectroscopy, Fluorescence Spectroscopy or particle counting such as NTA system. NPs integrity will be studied with transmission electron microscopy (TEM) and DLS.

Once biochemically characterized GelMA-NPs, these will be used to deliver bioactive compounds identified through proteomics data as subsequently described.

To validate the developed bio-ink in in vitro craniofacial disease models, patients with craniofacial malformations undergoing cranial surgery will be selected and enrolled in the study. Specifically, paediatric patients affected by craniosynostosis and other inborn defects, trauma or brain tumors undergoing cranial surgery will be enrolled. Adult patients undergoing craniofacial surgery will be also selected and enrolled for craniofacial trauma and tumors. To this purpose, cranial bone tissue specimens derived from both paediatric and adult patients will be obtained as surgical waste during craniectomy or cranial vault remodeling upon obtaining the signed informed consent (by parents in case of children). For each patient, the waste tissues will be randomized into three aliquots. Of these, 1 aliquot will be collected in cell growth medium for seeding bone tissue samples into culture plate to isolate mesenchymal stromal cells (MSCs). Thereafter, MSC will be expanded up to the 3rd culture passage and collected in biobanking infrastructures with associated anonymized clinical data and optimized tracking system for subsequent analysis as follows. GelMA-based bioink NP release and biocompatibility will be analyzed using MSC, through functional assays to: evaluate focal adhesion dynamics and study cell adhesion to the gelified bioink by immunofluorescence assay. Cell behaviors and viability will be measured in time course experiments using a live cell imaging system (Incucyte Live Cells Analysis systems).